NO129093B - - Google Patents

Description

Triiodobenzoic acid derivatives for use as

X-ray contrast agents and for use in solutions with high specific gravity for use in biological separation.

The present invention relates to new triiodobenzoic acid derivatives for use as X-ray contrast agents and for use in solutions with a high specific gravity for use in biological separation.

When X-rays make relatively large areas of the human body visible, e.g. the cardiovascular system, or the space containing the cerebrospinal fluid, large amounts of high-concentration X-ray contrast agents must be injected to provide sufficient density in the area concerned. Consequently, the toxicity of the contrast agent at high concentrations is of great importance. To make Ise visible in the cardiovascular system, a large number of compounds have been proposed as contrast agents, and although many of these have been used with good results, their toxicity, although it is often quite small, entails a number of unwanted side effects. When visualizing the space containing the cerebrospinal fluid, the highly concentrated compounds used for cardiovascular visualization are often far too toxic, which will be explained below.

The ideal contrast agent for the subarachnoid space has not yet been found. Gases and oils have been proposed, but these have many disadvantages. The water-soluble iodomethane sulphonate is the principle common contrast agent for this area, it has a high density and is absorbed quite quickly. However, this substance is far from ideal, when highlighting the different zones in the subarachoid space it is usually used for radiculography, but the simultaneous use of an anesthetic agent is necessary and it is too toxic for cisternography, ventriculography and cervical or thoracomyelography.

As indicated above, the iodinated compounds commonly used in the cardiovascular system are usually chosen because of their high solubility in water, solubilities of the order of 100 g/100 ml being common. In order to achieve water solubility, compounds which carry an acidic group have usually been chosen, e.g. a carboxylic acid group or a sulphonic acid group, as their alkali metal salts and certain amine salts are often extremely easily water-soluble and even though several of the commercially used contrast agents of this type exhibit an extremely low degree of toxicity. intravenously, their use at high concentrations has been shown to lead to undesirable side effects when they precipitate in the cerebrospinal fluid. In addition to the toxic effects caused by the ions, it has been demonstrated that these side effects are partly due to osmotic imbalance produced by injecting very large concentrations of dissolved material into the body fluids.

The osmotic pressure of a solution of a chemical compound is usually approximately directly proportional to the sum of the concentrations of the various molecular or ionic substances present. A water-soluble salt, e.g. the sodium salt of an iodinated acid will normally be almost completely ionized and the osmotic pressure will be proportional to the concentration of both the anion and the cation. The total concentration of ionic substances will thus be approximately twice the concentration of the salt considered as a single non-ionized substance. In contrast, one would expect that the osmotic pressure of a non-ionized compound, i.e. a compound that is substantially non-ionized in aqueous solution, is approximately proportional simply to the molarity of the compound present, i.e. approximately half the value for an analogous ioni-

serted compound that exhibits two ionized moieties.

Our investigations of the osmotic pressure have actually

shown that the group of non-ionized compounds according to the invention which is defined more carefully in the following, exhibits values for the osmotic pressure which are considerably lower than those which one would expect on the above-mentioned basis.

We have now examined a larger number of non-ionized iodinated compounds and have been able to determine that, in the particularly sensitive area of cerebrospinal visualization, the toxicity of their concentrated, aqueous solutions is generally very significant.

substantially lower than the toxicity of the best ionic compounds proposed for use for such a purpose and is generally lower than the toxicity of solutions of the corresponding ionic compounds

ized connections»

Many of the compounds further exhibit intravenous toxicities that are markedly lower than the toxicity of the best commercial vascular contrast agents, and are consequently of further value in cardiovascular imaging.

Our investigations have further led us to that conclusion

that the iodinated compound should be an alkanol bearing at least one N-linked secondary or tertiary amide group. In addition, the molecule must have at least two hydroxyl groups.

According to the present invention, triiodobenzoic acid derivatives for use as X-ray contrast agents and for use in high specific gravity solutions for use in biological separation are provided, which have the general formula

12

where R and R , which can be the same or different, are hydrogen atoms or alkyl groups which are optionally substituted with hydroxy, aliphatic acyoxy and/or aldehyde groups, as they cannot both be hydrogen at the same time, and R<3> and R< 4>, which may be the same or different, are hydrogen atoms, acylamino groups of the formula NR 5 Ac where R <5> is a hydrogen atom or an alkyl group optionally substituted with hydroxy, aliphatic acyloxy and/or aldehyde groups, or an aliphatic acyl group and Ac is an aliphatic acyl group, acylaminomethyl groups of the formula

5 5

CH-NR Ac where R and Ac have the meaning stated above, or carbamoyl groups of the formula CONR 6 R 7 where R <6> and R <7> are hydrogen atoms or alkyl groups which are optionally substituted with hydroxy-, aliphatic acyoxy- and/or aldehyde groups, and corresponding binuclear compounds containing two ring structures of formula i bonded together by a common substituent, wherein any alkyl group, substituted alkyl group or aliphatic acyl group present contains 1-6 carbon atoms, and at least one N is present -hydroxyalkyl group and at least two hydroxyl groups in the molecule.

The hydroxyalkyl groups that are present can carry a single hydroxy group, as in the /3-hydroxyethyl group, or more than one hydroxy group, as in the dihydroxypropyl or tris-(hydroxymethyl)methyl group or in the polyhydroxyalkyl portion of hexoamines, pentosamines, and amino sugar alcohols, such as glucosamine or glucamines, e.g. N-methylglucamine, 1-glucamine or 2-glucamine. Other non-ionic substituents may also be present, e.g. the aldehyde group present in glucosamine, or one or more acyloxy groups.

Alkyl, hydroxyalkyl and aliphatic acyl groups such as

is present, contains 1-6 carbon atoms. Preferred alkyl groups thus include methyl, ethyl, propyl, butyl

and hexyl groups, the methyl group is preferred and an N-methyl substituent often increases water solubility„

Preferred acyl groups which may be O-linked or N-linked include acetyl, propionyl and butyl groups, the acetyl group being the most preferred.

Alkyl, hydroxyalkyl and acyl groups which are present - can also carry a further non-ionic iodinated hydrocarbon group which can carry further amide groups and thus can e.g. an alkylene, hydroxyalkylene or diacyl group derived from a divalent acid, be N-bonded at any end to identically iodinated hydrocarbons bearing amide groups.

A number of compounds according to the invention

that have been produced are listed in Table I below.

As stated above, the new non-ionic compounds according to the invention have shown distinctly low toxicity, particularly in intracerebral experiments. Table 2 below shows results obtained with a number of compounds according to the invention compared to previously known X-ray contrast agents. It will be seen that all the new compounds listed are much better than the known compounds in the intracerebral experiment.

Most of the compounds were so indifferent or inert that it was not physically possible to inject a sufficient amount of substance to kill 50% of the mice and in such cases only a minimum value can be stated for the LD50

A number of the new compounds are distinguished by a very high water solubility. The water solubility is shown in table 3 below.

It will further be seen that the compounds shown in Table 3 which exhibit 3 or more hydroxyl groups, except when a secondary amide or an ester group is present, are highly water soluble and these are generally preferred.

However, it should be noted that although high water solubility is for many purposes desired in an X-ray contrast agent, water solubility is not of decisive importance and compounds with a very low toxicity may be useful even if they are water insoluble.

As indicated above, the compound according to the invention has a low osmotic pressure compared to the usual ionic contrast agents and many of the compounds even show a lower osmotic value than one would expect. The observed osmosis in each case would be expected to be approx. 0.8 mol/kg, but the compounds 3, 31, 33, 42 and 11 listed in Table I have shown osmosis values of 0.47, 0.48, 0.61, 0.53 and 0.48 mol/kg at 37°C (300 mg J/ml). For the sake of comparison, the last two compounds in table 2, which are ionic compounds, show osmosis values of approx. 1.6 mol/kg.

The compounds most preferred as contrast agents in the myelographic area include compounds 6, 9, 10, 11, 12, 15 and 31 from Table I.

These compounds are highly water-soluble and are

possessing acceptable viscosity values are also useful as cardiovascular contrast agents and compound 9 in Table I above is particularly useful for this purpose as it exhibits a viscosity of 7.1 cp at 20°C and a concentration of 300 mg j/ml. However, the preferred compounds listed above in connection with the myelographic area are also very useful for cardiovascular imaging as a result of their exceedingly good tolerability.

The concentration of the X-ray contrast agent according to the invention in the aqueous medium for administration varies with the particular purpose of use. In general, lower concentrations are required for ventriculography than for myelography, while radiculography requires even lower concentrations. The preferred concentration and dosage ranges of the compounds for these three purposes are as follows:

The preferred concentration range for cardiovascular visualization is 150 - 450 mg J/ml. The amount of the contrast agent to be administered is preferably such that it will

remain in the system only for approx. 2 to 3 hours, although both shorter and longer periods of stay are normally acceptable. For cerebrospinal visualization, the active material must therefore be suitably introduced into ampoules containing 5 - 15 ml of an aqueous solution of the material. but for vascular visualization, larger amounts are used, e.g. 10 to 500 ml.

The new compounds according to the invention can be prepared in any suitable way.

1) A particularly suitable method comprises the reaction of a compound of the general formula

8 9

(where R and R which can be the same or different have

3 4 — the meaning given above for R and R , or are carboxyl groups) or an amide-forming derivative thereof, with a pre-12 12

bond of the formula HNR R (where R and R have the above an-

brought meaning). The preferred method for carrying out this reaction is to condense an acid halide, e.g. the bromide, or more advantageously, the chloride, with the compound of the formula NHR 1 R 2 .

The reaction is preferably carried out in an inert solvent, such as e.g. a cyclic ether, e.g. dioxane or tetrahydrofuran,

or an amide solvent, such as dimethylformamide or dimethylacetamide. A small excess of the amine is advantageously used and an acid binding agent may be present, e.g. an alkali metal carbonate or bicarbonate: or a tertiary amine, such as triethanolamine. A suitable excess of the amine can, however, serve as an acid-binding agent, or when it is a liquid, which. solvent.

For isolation of the carbamyl compounds, the residue from the evaporated reaction mixture can be dispersed in water and acid of formula II regenerated by hydrolysis of the acid halide can be dissolved by addition of alkali. When it is desired to: remove the acyl groups used to protect hydroxyl groups or NH groups during the formation of the acid halide, the treatment with alkali can be carried out at an elevated temperature, e.g. 50-60°C. The less water-soluble carbamyl compounds can be separated from such aqueous solutions. When it comes to more water-soluble carbamyl compounds, however, the separation from inorganic compounds entails

salts, e.g. such as are formed between the acid binding agent and the liberated hydrogen halide, or such as are formed by neutralization of the acid halide, difficulties. Under such circumstances, the water-soluble carbamyl compound can be isolated, e.g. by phenol extraction.

The residue from the reaction mixture can thus be dissolved in water and, if necessary, treated with alkali, as described above, after which the solution is made acidic, e.g. by adding a mineral acid, such as hydrochloric acid, e.g. to a pH of approx. 1 and extracted with phenol. The phenol extraction is preferably carried out with a number of separate extractions, e.g. 3 or 4, and the phenol extracts are combined. Each phenolic extract is advantageously 1/10 to 1/5 of the aqueous volume. The phenol is preferably 90% aqueous phenol. The combined phenolic extracts are then washed several times, e.g. 3-5 times, with water to extract remaining inorganic salts, and approx. 2-3 parts by volume of ether. The phenol/ether solution is then extracted with water, preferably several times, e.g. 3-5 times, using 1/10 volume of water for each extraction. The aqueous solution is then washed with ether to remove residual phenol and evaporated to dryness to give the desired contrast agent. It is also possible to purify water-soluble contrast agents by bringing the reaction solution into contact with ion exchange resins, e.g. cation exchange resins to remove cations from the acid binder and/or anion exchange resins to remove the acid from the hydrolyzed acid halide.

The products can also be isolated by extraction of the reaction mixture, for example with cyclic ethers, such as tetrahydrofuran or dioxane.

The acid halides of the acids of formula II, which are themselves new compounds, can be prepared by reacting the acid with a halogenating agent, such as thionyl chloride or bromide, phosphorus pentachloride or bromide or phosphorus oxychloride or oxybromide. An inert solvent, e.g. a cyclic ether, such as dioxane or tetrahydrofuran, or a hydrocarbon solvent, such as e.g. benzene or toluene can be used or an excess of the reagent can serve as the reaction medium. When free NH and/or OH are present in the starting acid, however, these can react with the halogenating agent and in such cases they are preferably protected. Acylation is the most appropriate form of protection, as both N-acyl and O-acyl groups can be added simultaneously during the reaction with an acylating agent, such as e.g. an acyl halide or an anhydride. The acyl groups are preferably aliphatic acyl groups, such as propionyl, or more advantageously, acetyl groups. Hydroxyl groups on sugar or sugar alcohol residues can also be protected, e.g. by ketal formation.

A particularly suitable class of protecting groups are trialkylsilyl groups, e.g. the trimethylsilyl group. All the free hydroxyl groups in the hydroxyamine reagent can be suitably protected simultaneously in this way, e.g. by reaction with trialkylsilyl halide, e.g. the chloride, preferably at low temperatures, e.g. 0-20°C, to avoid formation of N-derivatives. Tertiary amines such as pyridine are particularly suitable solvents and also serve as acid binders. An inert solvent such as an ether may also be present.

As indicated earlier, these protective groups may remain during the amide formation, but can then be removed by hydrolysis.

The trialkylsilyl protecting groups can then be removed by hydrolysis with dilute acid, e.g. an aqueous alcoholic solution of hydrochloric acid at a pH of 2-3. It is also possible to form acid half-genides by halogenation of acids of formula II which have no hydroxyalkyl groups, and to introduce these groups then e.g. in the methods described below.

2) Another method involves the reaction of a compound with the general formula III

12 4

(where R , R , R and. Ac have the meanings given above)

with an alkylating, acyloxyalkylating or hydroxyalkylating agent. The "alkylating" agent can e.g. be a reactive -mono-ester derivative of an alcohol or polyol, e.g. a halide, e.g. a chloride or bromide, or a sulfate or a hydrocarbon sulfonate. For introducing a hydroxyethyl group, 2-chloroethanol is a suitable agent. For the introduction of a methyl group, dimethyl sulfate is a suitable agent. The reactive derivative is preferably reacted with the acylamide starting material under basic conditions, e.g. in an aqueous alkaline medium, such as e.g. contains an alkali metal hydroxide, such as sodium or potassium hydroxide, or in a non-aqueous medium, e.g. in an alkanol, such as methanol or ethanol, the base is conveniently an alkali metal alkoxide, such as sodium methoxide. It is also possible to react the acylamide compound with an epoxide, e.g. ethylene oxide, propylene oxide, glycide, etc., advantageously in neutral alcoholic solution.

3) Reaction of .an iodinated amide as indicated in 2) above and

which exhibits an NH group, e.g. a carbamyl compound of formula III, with an allylating agent, e.g. a reactive derivative of. an allyl alkphol, e.g. allyl chloride or bromide, to introduce an N-allyl group which is then subjected to oxidation of its double bond, e.g. using a permanganate oxidizing agent, to form a glycol group, thereby forming a non-ionic amide with a. N-dihydroxyalkyl group, e.g. a compound of formula I in which is a dihydroxyalkyl group, such as a dihydroxypropyl group.

4) Another method involves the reaction of a compound of the formula

with an acylating agent followed by hydrolysis of any unwanted acyloxy groups that are formed during the reaction.

The acylating agent can, for example, be an acid anhydride (such as

can also serve as a solvent) together with catalytic amounts of a mineral acid, e.g. sulfuric acid or perchloric acid, or an acid halide preferably in a polar solvent, such as dimethylformamide or dimethylacetamide, acid halides are preferred due to the fact that smaller amounts of by-products are formed. The basic hydrolysis of the O-acyl group can e.g. is carried out using an aqueous alkali metal hydroxide, e.g. sodium hydroxide and the reaction is preferably carried out at room temperature. Depending on the acylating agent used, other products may also be formed that require separation. When an acyl anhydride, such as acetic anhydride, is used with concentrated sulfuric acid as a catalyst, a primary amino-

group often, partially become bis-acylated, although the bis-acylamino group is very easily hydrolyzed to acylamide under mild basic conditions. 5) Iodination of a benzoic acid amide which carries at least one N-hydroxylalkyl group and which has at least two hydroxyl groups in the molecule. The iodination can conveniently be carried out using iodine monochloride or a complex compound thereof, such as sodium iodine dichloride. The reaction is preferably carried out in an aqueous medium, advantageously at an acidic pH.

The benzoic acid amide used as starting material can carry other groups in the molecule, but must have all three 2-, 4- and 6-positions unsubstituted. The 3 and/or 5 positions can

3 4

e.g. bear the groups R and R as indicated above in connection with formula I, but at least one 4 iri NI^ group should preferably be present.

A 3,5-diamino-benzoic acid amide which carries an N-hydroxyalkyl group and which has at least two hydroxyl groups can thus e.g. is made to react with an iodination reagent whereby iodine atoms can be introduced in the 2-, 4- and 6-positions, the free -groups are then transferred to avylamino groups with acylation, e.g. using acylating agents such as acid anhydrides or halides.

The 3,5-diamino-benzoic acid amide can be prepared by reduction of the corresponding 3,5-dinitro-benzoic acid amide or the 3-amino-5-nitro-benzoic acid amide, using reagents which serve to reduce a nitro group to an amino group, f .ex. catalytic hydrogenation, e.g. using a palladium catalyst, hydrazine and Raney nickel, or reaction with a source of sulfite, bisulfite or dithionite ions, e.g. sulfur dioxide or an alkali metal sulphite, bisulphite or dithionite in an aqueous medium, to give a sulphamino group, NHS0 3 H followed by hydrolysis to give an NI^ group (Piria reaction) e.g.- using a mineral acid such as hydrochloric acid. However, the sulfamino compound can, if desired, be reacted with an alkylating, hydroxyalkylating or acyloxyalkylating agent before the hydrolysis, whereby an alkylamino-, hydroxyalkylamino- or acyloxy-

alkylamino group.

The starting amide compound can be prepared by a method analogous to that stated above under (1).

The aforementioned iodination reaction can of course be carried out on free benzoic acids or reactive derivatives thereof, such as halides or esters, to provide starting materials for reaction (1).

The compounds of formula I, and especially those where R<3> means the group NR 5AC are subject to a number of different types of isomerism as explained below. The invention encompasses all such isomeric forms. Referring to the following formula

and the bonds which in this are given the numbers 1-4, one can distinguish between the following formulas for isomerism:

(a) Exo-endo isomerism due to a restricted rotation

of the N-CO bond (3) caused by steric hindrance from the neighboring large iodine atoms. These isomers tend to be in equilibrium in solution, but are sufficiently stable to be separated by thin layer chromatography, the glucoamide of N-methyl-3,5-bis-acetamide-2,4,6-triiodobenzoic acid consists e.g. of approx. 20%

of the endoform and 80% of the exoform. For such an isomerism to exist, R 5 must not be hydrogen.

(b) Cis-trans isomerism due to a restricted rotation

of bonds (1) and (2), is again caused by steric hindrance from the neighboring iodine atom. For this type of isomerism to

12 5

could be present, it is necessary that none of > R , R and R is hydrogen. While bond 2 does not seem to allow any rotation, the equilibrium of bond 1 seems to be more easily accessible and so far it has not been possible to separate cis-trans isomers.

(c) Syn-anti isomerism due to restricted rotation of the C-N bond (4). Naturally, R 1 and R 2 must be different and not hydrogen. The resistance to rotation of the bond (4)

is similar to that of bond (3), but so far no chromatographic separation has been possible.

o 12

When the group -NR R is the residue of a sugar amine, two further types of isomerism exist, namely: (d) Geometric isomerism of the hemiacetal bond in the cyclic sugar form. Mutarotation is possible so that although one form may be in excess when the amide is crystallized under neutral or basic conditions, acids lead to a proton-catalyzed equilibrium. Accordingly, when optical rotation is determined to characterize a sugar amide, the product should preferably first be equilibrated in acid to obtain a characteristic value which is not dependent on the presence of an excess of a hemiacetal isomer. (e) Optical isomerism due to the optical properties of the sugar amine. The D forms of the sugar amine have been used in the execution of the present work, but L forms can be used in a similar way. This type of isomerism naturally always exists when an asymmetric carbon atom is present and the sugar-alcohol amides therefore exist in the D or L form. The D-forms have also been particularly used in this case.

When the group NR 1 R 2 originates from glucosamine, yet another type of isomerism is possible, namely: (f) Glucose-mannose epimery. Glucosamides can undergo epimerism at the carbon atom adjacent to the aldehyde group present in the open chain form, which always coexists in equilibrium with the cyclic form. This epimerization is catalyzed by hydroxyl ions and forms the corresponding mannoside. In the synthesis of glucosamides under alkaline conditions will therefore usually

a quantity of mannoside be present in the starting reaction mixture and can be separated by thin-layer chromatography. However, for the practical application of the X-ray contrast compound, such separation is not necessary.

The acids of formula II are, in many cases, known compounds. Others are described in Belgian patent no. 734257. The carbamyl compound of formula III can actually be obtained for the corresponding acids by the above-mentioned method (1).

The highly water-soluble compounds according to the invention can, as a result of their large molecular weight, be dissolved to give solutions with a very high specific gravity. Such solutions are particularly useful in biological work techniques during which cells are handled in solutions of high specific gravity, e.g. by centrifugation or differential flotation because their low osmosis reduces the osmolysis of the cells that occurs when concentrated solutions of salts are used for this purpose.

Manufacture of

(a) Starting materials

(1) 3- amino- 5- N- methylacetamide- 2, 4, 6- triiodobenzoyl chloride

3-amino-5-N-methylacetamide-2,4,6-triiodobenzoic acid (586 g,

1.0 mol) was suspended in thionyl chloride (596 ml) and reacted with stirring at 70°C for 16 hours. Excess thionyl-

chloride was distilled off in vacuo, the residue dissolved in chloroform (2500 mL) cooled in an ice bath, washed with ice water (3 x 100 mL), saturated NaHCO 3 solution (3 x 100 mL), 2n Na 2 CO 3 solution (2 x 100 mL)

and finally with water (3 x 100 ml). After drying with CaCl2, the chloroform was distilled off and the residue dried in vacuo.

Yield: 522 g (91%), m.p. 145-160°C. A sample was recrystallized from ethyl acetate. Sm.p. 181-205°C. Found: Cl 5.37, calculated for C10H8C1J3N20: Cl 5.88. (2) 3-amino-5-N-methylacetamide-2,4,6-triiodobenzoyl chloride prepared using PCI,..

3-amino-5-N-methylacetamide-2,4,6-triiodobenzoic acid (58.6 g,

0.1 mol) was suspended in toluene (50 mL) and benzene (25 mL).

The benzene was distilled off to remove traces of water. Phosphorus pentachloride (20.8 g, 0.1 mol) was added with stirring at 40° and then heated to 70° and stirred for 16 hours. The new crystalline compound precipitated before all the starting material had dissolved. The reaction mixture was stored at -20° before filtration.

Yield: 52.9 g (87%). Found: Cl 5.78, calculated for C12H10<C>1J3N2°3: Cl 5.88.

(3) (a) 3- diacetylamino- 5- N- methylacetamide- 2, 4, 6- triiodobenzoyl chloride

The acid chloride from Preparation 2 (80 g) was suspended in acetic anhydride (400 ml) and heated to 60°C. Concentrated H 2 SO 4 (0.16 mL) was added with stirring and the reaction mixture was

stirred at 100°C for 2 hours and at room temperature overnight. The filtered product was suspended in glacial acetic acid, filtered again and dried. Yield: 77 g (85%), m.p. 255-260°C. Recrystallized from dioxane, the compound melted at 261-265°C. Found: C 24.84, H'2.12, N 4.10, Cl 5.2. Calculated for C^H^CIJ^O^ C 24.43,

H 1.76, N 4.07, Cl 5.15.

(b) When the acetylation was carried out with crude acid chloride and

at room temperature only one acetyl group was introduced. Yield: 66%, m.p. 238-240 C (tetrahydrofuran).- Found: Cl 5.41. Calculated for C12H10C1J3N2°3: Cl 5.49.

(4) 3-N-(/3-acetoxyethyl)-acetamide-5-N-methylacetamide-2,4,6-triiodobenzoic acid

3-N-(β-Hydroxyethyl)-acetamide-5-N-methylacetamide-2,4,6-triiodobenzoic acid (268 g, 0.4 mol) was added portionwise to dry pyridine (500 mL) with stirring. The solution was heated to 50°C and acetic anhydride (80 mL, 0.8 mol) was added dropwise over 30 minutes. The stirring was continued for 1 hour, after which the pyridine was distilled off in vacuo. The remaining oil was dissolved in water (1000 mL), treated with bone charcoal at room temperature, filtered and the O-acylated product was precipitated with 6n HCl to pH 1.5. The acid is filtered after stirring at room temperature for 16 hours and dried in a vacuum at 70°C. Yield: 235.4 g (82.5%), m.p. 194-199°C. Recrystallized from dioxane m.p. 199-201°C. Found: C 2 6.84, H 2.54, N 4.01. Calculated for C,,H,_J.N_0,: C 26.91, H 2.40, N 3.92.

16 17 3 2 6

Footnotes to Table 5:

Compound properties in the stated preparation,

a Original starting material prepared by N-methylation of 3-butyric amide-2,4,6-triiodobenzoic acid according to British

patent No. 987,796.

b The corresponding 3-diacetylamino-2,4,6-triiodobenzoic acid was prepared from 3-acetamide-2,4,6-triiodobenzoic acid by treatment

with moist acetic anhydride at 80°C.

c The corresponding O-acetylated acid was prepared by acetylation of 3-N-methylacetamide-5-N-(2,3-dihydroxypropyl)-acetamide-2,4,6-triiodobenzoic acid by a method analogous to preparation 4.

The starting materials shown in Table 5 were used in the preparation of the X-ray contrast agents listed in Table 1. Table 6 indicates the methods used and experimental details with reference to the following preparation methods showing the particular method used. The method described in some of the examples can of course vary somewhat with respect to unimportant features.

Method of manufacture

(b) Compounds in Table 1

(5) N-(3-N-methylacetamide-2,4,6-triiodobenzoyl)-glucamine

The acid chloride from Preparation 1 (12 g, 0.02 mol) was dissolved in dioxane (120 ml). To the solution was added water (25 mL) and NaHCO 3 (1.9 g, 0.022 mol). Glucamine (4.0 g, 0.022 mol) was added in portions and the reaction mixture was stirred at room temperature for 24 hours. The solution was evaporated to dryness in vacuo, the residue dissolved in water (500 mL), filtered clear and passed through an Amberlite IR 120 H+ ion exchange column. The effluent was evaporated to dryness in vacuo and a white crystalline residue was obtained. Yield: 11.7 g (80%), m.p. 100-120°C. The product was recrystallized from isopropanol (treated with bone charcoal in the solution), dissolved in water and treated with bone charcoal at 100°C for 20 min. The water was distilled off in vacuum and the white residue dried in vacuum at 70°C, m.p. 120-130°C.

Found: C 26.34, H 3.05, N 3.95, J 51.4

Calculated for C13H21 : C 26.17, H 2.83, N 3.82, J 51.87

(6) N-(3-diacetylamino-5-N-methylacetamide-2,4,6-triiodobenzoyl)-N-methylglucamine

The acid chloride from preparation 3(a) (41.4 g, 0.06 mol) was

dissolved in dioxane (750 ml). Water was added to the solution

(150 mL) and KHCO 3 (6.6 g, 0.066 mol) with stirring at room temperature. N-methylglucamine (12.9 g, 0.066 mol) was added in portions. After stirring for 20 hours, the solution was evaporated to dryness in vacuo, the residue dissolved in water (400 ml) at 50°C, filtered clear, pH adjusted to 1 and treated with bone charcoal at room temperature for 16 hours, filtered, the filtrate was extracted with phenol (4 x 50 ml), the phenol is washed with water (4 x 40 ml) and diluted with ether (600 ml), the phenol/ether was extracted with water (4 x 50 ml)

and the combined aqueous layers were washed with ether (3 x 30 mL) and evaporated to dryness in vacuo.

Yield: 38.3 g (75%), m.p. 115-126°C. The product was recrystallized from isopropanol (treated with bone charcoal in solution), dissolved in water, treated with bone charcoal at 60°C, the filtrate evaporated to dryness and the purified product dried in vacuum at 70°C. Sm.p. 155-165°C. Found: J 44.2, calculated for c2iH28J3N209: J 44.93

(7) N-(N- methyl- 3, 5- diacetamide- 2, 4, 6- triiodobenzoyl)- N- methyl-

glucamine

The triacetyl derivative from preparation 6 (29 g) was hydrolyzed by dissolving in water at 60° and adding 2N sodium hydroxide solution dropwise at pH 10-11 until the pH is stabilized at 10.8.

At room temperature, the pH was adjusted to 1, the solution treated with bone charcoal for 1 hour, filtered and extracted with phenol etc. as described in preparation 6 (4 x 40 ml phenol, 3 x 50 ml water, 350 ml ether, 4 x 50 ml water and 2 x 25 ml ether). 25.8 g (91%) were isolated, m.p. 110-130°C. The product was purified as described in Preparation 6. M.p. 165-170°C. [ a ]J° -4.5 (c 10% in 0.1 n HC1) Found: C 28.38, H 3.53, N 5.49, J 47.1

Calculated for C^H^J^Og: C 28.34, H 3.26, N 5.22, J 42.29

(8) N-[3-N-(/3-hydroxyethyl)-acetamide-5-N-methylacetamide-2,4,6-triiodobenzoyl]-N-methyl-2,3-dihydroxypropylamine

3-N-(β-acetoxyethyl)-acetamide-5-N-methylacetamide-2,4,6-triiodobenzoyl chloride (28.3 g, 0.04 mol) was dissolved in dioxane (280 mL). Water (60 mL) and KHCO 3 (4.4 g, 0.044 mol) were added with stirring. To this solution was added N-methyl-2,3-dihydroxy-propyl-

amine (4.63 g, 0.044 mol) dissolved in dioxane (5 mL) was added dropwise at room temperature over 15 minutes. Stirring was continued for 16 hours; The solution was evaporated to dryness, the residue dissolved in water (-200 ml), filtered clear, the solution heated to 60°C and 2N sodium hydroxide solution was added in

drops (pH 10-11) until the pH became stable at 10.5. At pH 1, the solution was treated with bone charcoal for 1 hour at room temperature. The filtrate was evaporated with phenol as previously described. The obtained aqueous solution was treated with bone charcoal at room temperature for 16 hours and evaporated to dryness. Yield: 25.5 g (84%), m.p. 140-145°C. Found: C 28.70%, H 3.75% N 5.45%, J 50.3% Calculated' for C18H24J3N306: C 28.51, H 3.19, N 5.54, J 50.2. (9) 3,5-bis[N-(2',3'-dihydroxypropyl)-N-methylcarbamoyl]-N-.

(2'- hydroxyethyl)- 2, 4, 6- triiodoacetanilide

3,5-bis[N-(2',3'-dihydroxypropyl)-N-methylcarbamoyl]-2,4,6-triiodacetanilide (3.9 g, 5 mmol) was dissolved in water (8 mL) and then 5N sodium hydroxide (3 mL) and 2-chloroethanol (0.67 mL, 10 mmol) were added. After two days at room temperature, the solution was acidified with 6N hydrochloric acid and evaporated to dryness in vacuo. The residue was dissolved in water (15 ml) and extracted with phenol (4x5 ml), the combined phenol extracts were washed with water and diluted with ether (60 ml). This phenol-ether mixture was extracted with water (4 x 10 ml) which was finally washed with ether. The aqueous solution was then evaporated in vacuo

dryness. Yield: 1.6 g (40%). Sm.p. 146-159°C.

(Found: C 29.26, H 3.67, J 46.2, N 5.08.

Calculated for C^H^J^Og: C 29.32, H 3.45, J 46.47, N 5.13).

continued

Additional manufacturing methods

(10) N-(3<5-diacetamide-2,4,6-triiodobenzoyl)-N-methylglucamine

(a) N-(3,5-dinitrobenzoyl)-N-methylglucamine

N-methylglucamine (21.4 g, 0.11 mol) was suspended in

DMF (200 mL). Triethylamine (11.5 g, 0.11 mol) was added. To

to this suspension was added at 4 with stirring 3,5-dinitro-benzoyl chloride (23.0 g, 0.1 mol) dissolved in dioxane (100 ml). The temperature rose to 10°. Stirring was continued at this temperature for 2 hours followed by 16 hours at room temperature. Triethylamine hydrochloride was filtered off and DMF distilled off from the filtrate. The residue, a light brown oil, was dissolved in water (200 mL),

pH adjusted to 1 and the solution extracted with phenol according to standard procedures. The obtained aqueous solution was treated with bone charcoal at room temperature for 24 hours and the filtrate evaporated to dryness in vacuo. The residue - a light brown oil -

was further dried in vacuo at 65°. Yield: 25.5 g (65%). IR spectrum showed a characteristic carbonyl absorption band at 1670-1620 cm<-1>.

Rf value: 0.55 - 0.6 (paper, n-BuOH : EtOH : NH 2 : H 2 O =4:1:2:1).

(b) N-(3,5-diamino-2,4,6-triiodobenzoyl)-N-methylglucamine

The product from step (a) (7.78 g, 0.02 mol) was dissolved in methanol (150 ml) and hydrogenated at room temperature and 3 kg/cm 2 during 16 hours. Catalyst 1 g 5% Pd/C. The catalyst was filtered off, the filtrate treated with bone charcoal at pH 2 and the methanol distilled off in a vacuum. The residue - a pale yellow oil - was dissolved in water, acidified to pH less than 1 and treated with bone charcoal at room temperature. A paper chromatogram (n-BuOH : EtOH : NH^ : H 2 O = 4:1:2:1) showed the desired product with an R^ value of 0.14.

A 3.75 n NaJCl 2 solution (17.6 ml, 3.3 eq.) was added to the filtrate over 15 minutes. The iodinated product separated as a dark brown oil. The reaction mixture was allowed to stand at 3°, the supernatant liquid was decanted from and. the oil dried in high vacuum at room temperature. The oil crystallized during this process. Yield: 7.5 g (53%), m.p. 110°.

(c) N-(3,5-diacetamide-2,4,6-triiodobenzoyl-N-methylglucamine

The product from step (b) (3 g, 4.2 mmol) was suspended in acetic anhydride (30 mL). After stirring at room temperature for 1 hour, concentrated H 2 SO 4 (0.3 mL) was added. All material was dissolved. Stirring was continued for 16 hours before the acetic anhydride was distilled off in vacuo. The oily residue was dissolved in 1N sodium hydroxide solution (100 ml), acidified to pH 1 with 6N hydrochloric acid, treated with bone charcoal at room temperature and the filtrate extracted with phenol. The resulting aqueous solution was evaporated to dryness in vacuo and the residue - a greyish crystalline product - was dried in vacuo at 70°. Yield: 0.9 g (27%), m.p. 145-165°. Recrystallized from methanol, m.p. 155-167°.

This compound shows identical IR spectrum and chromatographic data when compared to compound 49

in Table 6 which was prepared by reacting starting material 13 with N-methyl-glucamine.

(11) N-(2,4,6-triiodobenzoyl)-N-methylglucamine

(a) 2, 4, 6-triiodobenzoyl chloride

2,4,6-triiodobenzoic acid (15 g) was suspended in thionyl chloride (75 ml) and refluxed. 30 minutes after initiation, the material was dissolved, the reaction solution was cooled and evaporated in vacuo. The residue was dissolved in hot benzene (40 ml), cooled to room temperature, filtered and the filtrate evaporated in vacuo. Yield: 13.6 g.

I.R. (KBr): 1785 cm" (-C0C1).

(b) N-(2,4,6-triiodobenzoyl)-N-methylglucamine

2,4,6-triiodobenzoyl chloride (13.6 g, 26.2 mmol) was dissolved

1 DMF (30 ml) and cooled in ice water. Potassium carbonate (4.0 g,

29 mmol) was added with stirring and then N-methyl-glucamine (5.65 g, 29 mmol) was added over 90 minutes. After 4 hours the temperature was allowed to rise to room temperature. After 2 days, the suspension was filtered, and the filtrate evaporated in vacuo. The residue was dissolved in water (75 ml) and the pH adjusted to approx. 0.5 - 1.0 with hydrochloric acid. The acidification led to a refinement of a gummy substance which crystallized when treated with methanol. Finally, the product was suspended in water (25 ml) for 2 hours. Yield: 11.7 g (66%). Sm.p. 178-190°.

IR (KBr): 1620 cm"<1> (CON), broad band at 3300 cm<-1> )OH).

(Found: C 24.17, H 2.75, J 57.0, N 2.26

Calculated for C14H1Q<J>3<N>06: C 24.84, H 2.68, J 56.28, N 2.0<7>.

Production 12

3, 5- bis-[ N-(2, 3- dihydroxypropyl)- N- methylcarbamyl1- 2, 4, 6- triiodo-acetanilide (compound 9)

Acid chloride No. 11 from Table 4 (3.2 g, 0.005 mol) was dissolved in dimethylformamide (10 ml) and cooled in ice-water. Potassium carbonate (1.52 g, 0.011 mol) was added with stirring. A solution of 3-methylamino-propanediol-2,3 (1.16 g, 0.011 mol) in dimethylformamide (85 mL) was added over 15 minutes. After 4 hours the temperature was allowed to rise to room temperature and stirring continued for a further 20 hours. The mixture was filtered, the filtrate evaporated to dryness in vacuo and the residue extracted with phenol in the usual manner. The resulting aqueous solution was evaporated to dryness in vacuo to give 1.5 g (39%) of the desired product. Sm.p. 60-75°C (decomposition). This product was dissolved in methanol (10% solution) and the solution diluted with isopropanol (2 times the volume), decanted from the precipitated colored impurities and evaporated to dryness in vacuo. The residue was dissolved in water, treated with bone charcoal, evaporated to dryness in vacuo, redissolved in water, treated again with bone charcoal and the filtrate evaporated to dryness in vacuum. Sm.p. 135-159°. Found: C 28,29,

H 3.52, N 5.60, J 48.5. Calculated for <C>18<H>24<J>3<N>3<0>7<:> C 27.89,

H 3.12, N 5.42, J 49.11.

(13) N-(N- methyl- 3, 5- diacetamide)- 2, 4, 6- triiodobenzoyl)- glucosamine

Acid chloride from Preparation 3 (41.3 g, 0.06 mol) was

reacted with glucosamine and hydrolyzed as described for compound 10. Yield: 36.0 g (76%).

(14) N-(N- methyl- 3, 5- diacetamide- 2, 4, 6- triiodobenzoyl)- glucosamine ( method no. 2

N-Methyl-3,5-diacetamide-2,4,6-triiodobenzoyl chloride (32.3 g, 0.05 mol) was dissolved in DMF (300 mL) at 0 °C. Traces that were unsolved. Potassium carbonate (13.8 g, 0.1 mol) and glucosamine hydrochloride (10.8 g, 0.05 mol) were added. The suspension was stirred at 0° for 2 hours and then at room temperature for a further 20 hours. Potassium carbonate (2.76 g, 0.02 mol) and glucosamine hydrochloride (2.15 g,

0.01 mol) was added and stirring was continued at room temperature for 46 hours. Total reaction time: 68 hours.

Inorganic salts were filtered off and the filtrate evaporated to dryness in vacuo at 50-55°. The residue was dissolved in water

(200 ml), acidified with hydrochloric acid to pH 1 and treated with bone charcoal at room temperature for 16 hours. The aqueous solution was extracted with phenol as described above. The aqueous extract obtained (pH approx. 4) was treated with bone charcoal for 20 minutes at 80°. The faintly colored filtrate was evaporated to dryness in vacuo

and the residue is dried in vacuum at room temperature for 24 hours. Dividend; 31.0 g (78%). Temp. 165-230° (dec.). The crude product was mixed with 20 w/w percent filter aid (speedex) and extracted in a Soxhlet apparatus with tetrahydrofuran for 20 hours. Yield: 65%. The product was further purified by recrystallization from isopropanol, dissolved in water and treated with bone charcoal first for 3 hours at room temperature, then for 30 minutes at 80-90° and finally for 16 hours at room temperature. The colorless, aqueous solution was evaporated to dryness in vacuo and the white, crystalline residue was dried in vacuo first at room temperature and then at 70°. Sm.p„ 230° (splitn.).

= 18.0° (K = 10% in 0.ln HC1, in equilibrium with respect to mutarotation).

Thin layer chromatography (Silica F, n-BuOH : H2O : AcOH =

100 : 50 : 22) showed 15-20% endo isomer (Rf 0.44) and 80-85% exo isomer (Rf 0.68), confirmed by nuclear magnetic resonance.

(15) N-(3-acetamido-5-N-methylcarbamoyl-2,4,6-triiodobenzoyl)-N-(2-hydroxyethyl)-glucamine

3-acetamido-5-N-methylcarbamoyl-2,4,6-triiodobenzoyl chloride

(18.4 g, 29 mmol) was suspended in dimethylformamide (100 mL) and cooled in ice water. Then potassium carbonate (4.6 g, 33 m mol)

and N-(2-hydroxyethyl)-glucamine (8 g, 36 m mol) added. Blendin-

gen was vigorously stirred. After 4 hours the temperature was allowed to rise to room temperature. The mixture was stirred for two days and then felt-

correct. The filtrate was evaporated in vacuo to dryness, and the residue was dissolved in water (50 ml). The aqueous solution was then acidified with hydrochloric acid and extracted with phenol (4 x 25 mL). The combined phenolic extracts were washed with water, then diluted with ether (300 mL) and extracted with water (5 x 30 mL). Finally, the aqueous layer was washed with ether (4 x 20 mL) and evaporated to dryness in vacuo.

Yield: 18.9 g (79%).

The product was dissolved in 80 percent (volume/volume)

aqueous methanol (180 ml) and stirred with "Dowex" anion exchange resin 1x4 (5 g). The next day the pH was approx. 7.1. After filtration, "Amberlite" cation exchange resin IR 120 H (2 g) was added and the suspension was stirred for 2 hours. The pH of the solution was approx. 3.5.

Then "Dowex" anion exchange resin (2.5 g) was added. After

that the mixture had been stirred for several hours, the pH had risen to 6.1.

After filtration, "Amberlite" IR 120 H (2 g) was added, and mixed

The thing was stirred for approx. 2 hours. The pH dropped to 4.4. Then "Dowex" anion exchange resin (2 g) was added, the mixture was stirred for two hours and filtered (pH was about 6.1). The ion exchange resins were collected on a filter and washed with 80 percent (v/v)

lum) aqueous methanol. The filtered solutions were collected and evaporated to dryness in vacuo. Yield: 15.7 g. The product was recrystallized from isopropyl alcohol (120 ml). Yield: 11 g.

The product (10 g) was dissolved in water (10 ml) and extracted wood

times with a mixture of chloroform and butanol (60:40). The aqueous layer was then evaporated to dryness in vacuo. The product was redissolved in water and evaporated to dryness in vacuo twice. Yield: 9.0 g. M.p.: 178-190°C. (Found: In 45.8.

Calculated for CigH26I3N3Og: I 46.36.

Claims (8)

1. Triiodobenzoic acid derivatives for use as X-ray contrast agents and for use in solutions with a high specific gravity for use in biological separation, characterized in that they have the general formula 1 2 where R and R , which may be the same or different, are hydrogen atoms or alkyl groups which are optionally substituted with hydroxy, aliphatic acyoxy and/or aldehyde groups, since they cannot both be hydrogen at the same time, 3 4 and R and R , which may be the same or different, are 5 5 hydrogen atoms, acylamino groups of the formula NR Ac where R is a hydrogen atom or an alkyl group which is optionally substituted with hydroxy, aliphatic acyoxy-'Og/or aldehyde groups, or an aliphatic acyl group and Ac is an aliphatic acyl group, acylaminomethyl groups of the formula CH2NR 5 Ac where R 5 and Ac have the meaning stated above, or carbamoyl groups of the formula 6 7 6 7 CONR R where R and R are hydrogen atoms or alkyl groups optionally substituted with hydroxy, aliphatic acyloxy and/or aldehyde groups, and corresponding binuclear compounds containing two ring structures of formula I bound together by a common substituent, any alkyl group, substituted alkyl group or aliphatic acyl group present contains 1-6 carbon atoms and at least one N-hydroxyalkyl group and at least two hydroxyl groups are present in the molecule. 2. Connection as stated in claim 1, characterized in that it is N-(3-acetamide-5-N-methylcarbamoyl-2,4,6-triiodobenzoyl)-N-methylglucamine. 3. Connection as stated in claim 1, characterized in that it is 3,5-bis-[N-(2,3-dihydroxypropyl)-N-methylcarbamyl]-2,4,6-triodo-acetanilide. 4. Connection as stated in claim 1, characterized in that it is N-[3-N-(13-hydroxyethyl)-acetamide-5-N-methylacetamide-2,4,6-triiodobenzoyl]-glucosamine. 5. Connection as stated in claim 1, characterized in that it is N-(N-methyl-3,5-diacetamide-2,4,6-triiodobenzoyl)-glucosamine. 6. Connection as stated in claim 1, characterized in that it is N-[N,N'-di-(15-hydroxyethyl)-3,5-diacetamide-2,4,6-triodobenzoyl]-glucosamine. 7. Connection as stated in claim 1, characterized in that it is N-(N-methyl-3,.5-diacetamide- 2,4,6-triiodobenzoyl)-2-glucamine. 8. Compound as stated in claim 1, characterized in that it is N-(N-methyl-3,5-diacetamide-2,4,6-triiodobenzoyl)-D-glucamine.